Biologic sample collection devices and methods of production and use thereof

ABSTRACT

Collection devices and kits for biological sample collection include a biologic sample collection device having a hydrophilic swab matrix that includes a modified polycaprolactone (PCL). Methods of production and use thereof are also described herein. The biologic sample collection devices, kits and methods described herein are used to collect a biologic sample (e.g., blood, buccal cells, etc.) and to enable extraction of nucleic acids (e.g., DNA) from that biologic sample so that the nucleic acids can be analyzed (e.g., sequencing and subsequent analysis of DNA).

FIELD OF THE INVENTION

Collection devices, kits and methods related to biologic samplecollection and more particularly collection devices, kits and methodsrelated to nucleic acid sample collection using a hydrophilic swabmatrix are described herein.

BACKGROUND

Collection of biological samples for forensic identification, diagnosticapplications, and the like, is a critical first step in the process ofanalyzing such samples. Current practice in the art typically involvesswabbing an individual or area of interest containing deoxyribonucleicacid (DNA) with a cotton swab and subsequently extracting DNA frommaterial (in most cases nucleated cells) from the swab. While recentadvances in quality assurance allow for cotton swabs that are DNA-freeand of uniform quality, these advances do not address the basiclimitation of low DNA yield from cotton swabs, such as when used forforensic applications. Further, the quality of DNA extracted from cottonswabs can be inconsistent.

Accordingly, devices and methods for increasing DNA yield and qualityfrom a sample collection device are desired. Such devices and methodscould greatly increase the number of samples that could provide desiredinformation.

SUMMARY

Accordingly, one or more of the embodiments presented herein providecollection devices, kits and methods related to biological samplecollection and more particularly, collection devices, kits and methodsrelated to DNA sample collection using a hydrophilic swab matrix havinga synthetic component. Biologic sample collection devices are used totake samples from a variety of sources for applications such as forensicand diagnostic purposes involving identification of sample material bytesting of extracted nucleic acids (e.g., DNA). Testing can include, forexample, obtaining a genetic profile, and comparing it to referencesamples. Success of the identification process relies on the quantityand quality of the DNA extracted from the samples obtained on thecollection devices. A biologic sample collection device as describedherein takes up samples efficiently, and also releases a high yield ofDNA (which can be analyzed by one or more various methods or assaysknown in the art). Furthermore, a biologic sample collection device asdescribed herein lacks materials that impede steps in preparation of thesample or in the analysis of the sample or nucleic acids extractedtherefrom (e.g., detailed analyses performed to obtain DNA profilessuitable for unambiguous identification of the sample donor). Themanufacture and testing of innovative and improved biologic samplecollection devices and kits, as well as uses thereof, are the subject ofthe present application.

Herein are described biologic sample collection devices made fromsynthetic materials and from natural substances used either alone or inmixtures formulated to optimize one's ability to collect samples andrelease nucleic acids (e.g., DNA) from the collected samples. Samplesinclude any nucleic acid-containing biologic material such as all bodyfluids, nucleated cells obtained by various means and material taken inforensic investigations that may include cells shed in fingerprints.Samples may be those obtained directly from a subject (e.g., a humansubject) or those indirectly obtained from a subject (e.g., samples thathave been processed in some way prior to obtainment from the subject,samples left at a crime scene, etc.). In one example, the syntheticpolymer polycaprolactone (PCL) is used to form heads for swabs designedto collect samples for nucleic acid (e.g., DNA) analyses used foridentification of the sample donor (e.g., sequencing DNA from the sampleto identify the individual from whom the sample was obtained ororiginated). The configuration of the swab varies with its intended useand may include numerous sizes and shapes. In the experiments describedherein, yields of DNA obtained with the biologic sample collectiondevices described herein were shown to be superior to those obtained incontrolled comparisons using cotton swabs as a collection means.

The synthetic materials to be used in collection devices are in somecases not sufficiently absorbent or hydrophilic to give optimumcollection. Treatments to render the materials more hydrophilic andtests to assure success of the treatment are applied to collectiondevices. These may include changes in the manufacturing process or inthe composition of the material such as by copolymerization ofmaterials, addition of absorbent fibers in the manufacture, etc.Properties of the biologic sample collection device may also be changedby post-manufacturing treatment of the collection device. In oneexample, the PCL swab is treated under carefully controlled conditionswith an aqueous base, then neutralized, either by water washing orexposure to acid, and dried. Water uptake by the treated collector ismarkedly improved over the original non-treated product as shown byqualitative and quantitative measurements as described in the Examplesbelow.

Ease of operator use is aided by the assembly of the biologic samplecollector into its own plastic container. The entire assembly issterilized and rendered nucleic acid (e.g., DNA, RNA)-free by anysuitable means or process (e.g., exposure to ultraviolet radiation at anintensity and time that destroys any nucleic acids on the collector).The sterilization process also preferably kills any microorganismspresent. The plastic container keeps the collector sterile and DNA-freeprior to use. In one embodiment, the biologic sample collection deviceis housed within a plastic container and is operably connected to a capthat is used to seal the biologic sample collection device within theplastic container. In this embodiment, assembly of the biologic samplecollection device handle (referred to below as a carrier) into the capof the container allows samples to be taken by a user or operatorholding the biologic sample collection device by the cap; this avoidsthe user or operator touching the handle of the collector. After sampleacquisition, the biologic sample collection device is placed back intothe container where the fit of the cap prevents the swab tip fromtouching the interior (e.g., sides, tip) of the container to avoidlosing any sample by contact with the interior of the container.Although such a container will generally be plastic, any suitablematerial(s) (e.g., glass) can be used.

Release of DNA from standard cotton swabs has been shown to seldom yieldhigher than 20% of the actual amount present in the sample. Biologicsample collection devices prepared according to the methods describedherein routinely yield 80% of the sample's DNA as shown by controlledexperiments. An important factor in obtaining a high yield of DNA frombiological samples is the ability of the swab to release the materialand the extracted DNA into extraction reagent solutions. The fact thatthe hydrophilic, soluble PCL portion of the biologic sample collectiondevice (e.g., the PCL matrix) dissolves (is solubilized) in most of thecommonly used extraction solutions facilitates high yields of DNA.

Because the goal of the biologic sample collection device in someembodiments is to obtain a useable genetic profile of the individual orsubject from whom the sample was directly or indirectly obtained, thequality of DNA obtained and its suitability for subsequent testing isimportant. The biologic sample collection devices described hereinacquire sufficient sample and yield sufficient DNA to allow standardanalyses to be performed. This ability was tested using standardizedtests, which include DNA extraction with commonly used commercial kitsfollowed by amplification and analysis of areas of interest for geneticprofiles used in forensics applications. The biologic sample collectiondevices as described herein that were tested performed better thancomparison products such as cotton swabs in these tests.

In many applications, the collected sample is not analyzed immediatelyupon acquisition. An identification means to assure that the collectorand the sample can be processed without danger of losing the sampleinformation is typically attached to or included in a biologic samplecollection device as described herein. The use of bar codes or QR codesplaced on the device at the time of manufacture is one example of ameans of retention of sample identity, and in such embodiments, the datalinking the collector information with the sample is typically secure.Another embodiment of collecting a biologic sample involves the use ofan radio-frequency identification (RFID) tag that can be read withinreasonable proximity without having to scan all samples in a collectionand that can allow additional information about the sample to be addedat the time of collection. A passive RFID tag attached to the biologicsample collection device (or to the cap to which the biologic samplecollection device is attached per the description above) can be used,and guarantees that sample information and the collected sample cannotbe separated. Examples of sample information include date, time, person,location, collector, case number, and combinations thereof.

Accordingly, described herein is a biologic sample collection deviceincluding soluble and hydrophilic PCL coupled to a carrier, wherein atleast a portion of the PCL solubilizes when exposed to a nucleic acidextraction reagent. The PCL can be copolymerized with at least oneagent, e.g., acrylamide, polylactide, polyglycolide, polydioxanone, polyN-isopropylacrylamide, polyurethane, a polyester other than PCL, etc.Typically, the biologic sample collection device has been sterilized,and the PCL has been treated with a base having a pH greater than 8(e.g., NaOH, NaHCO₃, KOH, Na₂CO₃, and CA(OH)₂) and a neutralizing agentfor increasing hydrophilicity. In one embodiment, the PCL is coupled toa carbohydrate trehalose derivative group. In another embodiment, thePCL is coupled to at least one protein (e.g., antibody, silk, collagen,fibrin and elastin). The carrier can include one or more of: anidentifying label, a radio-frequency identification (RFID) tag, and abar code. The carrier can include a semi-rigid or rigid member (e.g., amaterial such as metal, plastic, polymer, wood, glass, or a combinationthereof). In some embodiments, the biologic sample is a human buccalsample. In some biologic sample collection devices, the PCL is containedwithin at least one aperture (e.g., two or more apertures) disposed inthe carrier. In such an embodiment, the at least one aperture can bedisposed at a first end of the carrier, and an identifying label can bedisposed at a second end of the carrier.

Also described herein is a kit for collecting at least one biologicsample. The kit includes: at least one biologic sample collection deviceincluding soluble and hydrophilic PCL coupled to a carrier, wherein atleast a portion of the PCL solubilizes when exposed to a nucleic acidextraction reagent; and packaging. A kit can further includeinstructions for use and at least one of: a nucleic acid extractionreagent; a nucleic acid labeling reagent; and a set of reagents fornucleic acid amplification. The packaging can include, for example, apolymer laminate or plastic container. In some embodiments, the kitincludes a plurality of biologic sample collection devices. In suchembodiments, each biologic sample collection device of the plurality ofbiologic sample collection devices can be individually packaged (or aplurality of biologic sample collection devices can be packaged withinone container or package). In one example, packaging includes: a firstcontainer including a rigid housing and a cap to which the biologicsample collection device is attached, the biologic sample collectiondevice positioned in the interior of the housing such that when the capis operably attached to the housing, the biological sample collectiondevice does not contact the interior of the housing; and a secondcontainer in which the first container and the biologic samplecollection device are contained. In one example, the first container isplastic and the second container is a plastic bag. In a kit, the PCL canbe contained within at least one aperture (e.g., two or more apertures)disposed in the carrier. The at least one aperture can be disposed at afirst end of the carrier, and an identifying label can be disposed at asecond end of the carrier. The PCL can be copolymerized with at leastone agent such as: polylactide, polyglycolide, polydioxanone,acrylamide, poly N-isopropylacrylamide, polyurethane, and a polyesterother than PCL. Typically, the biologic sample collection device hasbeen sterilized, and the PCL has been treated with a base having a pHgreater than 8 (e.g., NaOH, NaHCO₃, KOH, Na₂CO₃, and CA(OH)₂) and aneutralizing agent for increasing hydrophilicity of the PCL. The carriercan include one or more of: an identifying label, an RFID tag, a barcode, and an identifying label.

Further described herein is a method of producing a biologic samplecollection device as described herein. The method includes the steps of:providing a solution of solubilized PCL; submersing a carrier at leastonce into the solubilized PCL solution such that the solubilized PCLsolution coats the carrier; subjecting the solubilized PCLsolution-coated carrier to treatment with a base having a pH greaterthan 8 and a neutralizing agent under conditions that result in solubleand hydrophilic PCL coupled to the carrier; and sterilizing the solubleand hydrophilic PCL-coupled carrier such that the soluble andhydrophilic PCL-coupled carrier is free of nucleic acids, resulting in abiologic sample collection device including soluble and hydrophilic PCLcoupled to the carrier, wherein at least a portion of the PCLsolubilizes when exposed to a nucleic acid extraction reagent. Thecarrier can be submersed into the solution of solubilized PCL using afabricated mold. Examples of bases include NaOH, NaHCO₃, KOH, Na₂CO₃,and CA(OH)₂. Sterilizing the soluble and hydrophilic PCL-coupled carrierand rendering it free of nucleic acids can include, for example,exposing the soluble and hydrophilic PCL-coupled carrier to UVradiation. The method can further include copolymerizing the PCL with atleast one of: polylactide, polyglycolide, polydioxanone, acrylamide,poly N-isopropylacrylamide, polyurethane, and a polyester other thanPCL. The method can further include adhering to or coupling to thecarrier at least one of: an RFID tag, a bar code, and an identificationlabel.

Still further described herein is a method of extracting nucleic acidsfrom a biologic sample collected by a biologic sample collection deviceas described herein. The method includes the steps of: contacting thebiologic sample (e.g., human buccal sample) and the biologic samplecollection device with at least one nucleic acid extraction solutionunder conditions such that the PCL is solubilized or dissolved,resulting in a solution including nucleic acids separated from thebiologic sample collection device; and extracting the nucleic acids fromthe solution. The step of contacting the biologic sample and thebiologic sample collection device with at least one nucleic acidextraction solution can include immersing the biologic sample and thePCL in the at least one nucleic acid extraction solution (e.g., anextraction solution containing at least one proteolytic enzyme). In themethod, generally about 50% to about 95% of the nucleic acids from thebiological sample are extracted from the biological sample. The carriercan include an RFID tag and the method can further include obtaininginformation (e.g., date, time, person, location, collector, and casenumber) from the RFID tag.

Additionally described herein is a method for collecting a biologicsample. The method includes the steps of: contacting a biologic samplecollection device as described herein with a biologic sample such thatthe biologic sample is reversibly adhered to the PCL; contacting thebiologic sample collection device and the biologic sample with at leastone nucleic acid extraction reagent under conditions such that the PCLis solubilized and the sample is separated from the biologic samplecollection device; and collecting the separated sample. The method canfurther include extracting nucleic acids (e.g., DNA) from the separatedsample.

In another method of producing a biologic sample collection device asdescribed herein, the method includes the steps of: providing a solutionof solubilized PCL; forming at least a first flat sheet of solubilizedPCL; subjecting the at least first flat sheet of solubilized PCL totreatment with a base having a pH greater than 8 and a neutralizingagent under conditions that result in soluble and hydrophilic PCL thatsolubilizes when exposed to a nucleic acid extraction reagent; couplingthe at least first sheet of soluble and hydrophilic PCL to a carrier;and sterilizing the soluble and hydrophilic PCL-coupled carrier suchthat the soluble and hydrophilic PCL-coupled carrier is free of nucleicacids, resulting in a biologic sample collection device includingsoluble and hydrophilic PCL coupled to the carrier, wherein the solubleand hydrophilic PCL is a flat sheet, and at least a portion of thesoluble and hydrophilic PCL solubilizes when exposed to a nucleic acidextraction reagent.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

As used herein, “protein” and “polypeptide” are used synonymously tomean any peptide-linked chain of amino acids, regardless of length orpost-translational modification, e.g., glycosylation or phosphorylation.

By the term “gene” is meant a nucleic acid molecule that codes for aparticular protein, or in certain cases, a functional or structural RNAmolecule.

As used herein, a “nucleic acid” or a “nucleic acid molecule” means achain of two or more nucleotides such as RNA (ribonucleic acid) and DNA(deoxyribonucleic acid).

By the phrase “modified PCL” is meant any PCL that has been treated ormodified such that the hydrophilicity of the PCL is increased and/orsuch that one or more surface features of the PCL have been modified(e.g., chemical and/or physical modifications). Examples of surfacefeatures include texture (e.g., roughness, smoothness), holes, dimples,channels, protrusions and other irregularities. Any suitable treatmentmethods, including chemical or physical treatments, for increasinghydrophilicity and/or modifying surface features of PCL can be used. Forexample, PCL can be subjected to (treated with) a base (e.g. having a pHabove 8). Examples of bases include NaHCO₃ and NaOH.

As used herein, the phrase “soluble and hydrophilic PCL” meanspolycaprolactone (PCL) that has been treated in some manner to make itabsorb water and to become soluble in nucleic acid extraction reagents(e.g., DNA extraction solutions).

As used herein, when referring to a biologic sample collection device,the term “carrier” refers to any structure or implement to which PCL iscoupled, adhered, or disposed on or within. A carrier as describedherein assists man or machine in exposing PCL to a biologic sample, andsubsequent processing, e.g., hand held or “machine-held.” Examples ofcarriers include rigid and semi-rigid materials, such as wood, plastic,glass, rubber, and polymers.

By the phrase “nucleic acid extraction reagent” is meant any reagent(e.g., solution) that can be used to obtain a nucleic acid (e.g., DNA)from biological materials such as cells, tissues, bodily fluids,microorganisms, etc. An extraction reagent can be, for example, asolution containing one or more of: a detergent to disrupt cell andnuclear membranes, a proteolytic enzyme(s) to degrade proteins, an agentto inhibit nuclease activity, a buffering compound to maintain neutralpH, and chaotropic salts to facilitate disaggregation of molecularcomplexes.

As used herein, the term “copolymerized” refers to using two or moremonomeric units to form a polymer with inclusion of both in some random(e.g., AABABBBAABAAABBBBA) or defined order (such as, e.g., AAABAAABAAABor ABABABAB or ABAABAABAABAABAABA). For example, when referring to PCLthat is copolymerized with at least one agent such as, e.g., L-lacticacid, the copolymer formed is a poly caprolactide calledpoly-L-lactic-co-ε-caprolactone.

By the phrase “PCL composite solution” is meant PCL dissolved in asolvent. A PCL composite solution can be used to prepare a biologicsample collection device as described herein by filling in molds oradding between glass, plastic or metal plates, for example, to makesheets of PCL. Glacial acetic acid is one example of a solvent for PCLthat may be used.

The phrase “under conditions that result in soluble and hydrophilic PCLcoupled to the carrier” encompasses any suitable methods and steps fortreating PCL, a solubilized PCL solution, or a solubilized PCLsolution-coated carrier that contribute to an increase of hydrophilicityand/or solubility during preparation of a biologic sample collectiondevice as described herein. Conditions may include, for example,treatment of a solubilized PCL solution (e.g., a solubilized PCLsolution-coated carrier) with a base prior to neutralization, one ormore drying steps, one or more washing steps, etc.

As used herein, the phrase “flat sheet of solubilized PCL” means agenerally flat and thin (e.g., about ⅛ to about ⅜ inches thick) piece orportion of solubilized PCL. A flat sheet of solubilized PCL can be ofany suitable shape, including, for example, square or rectangular.

By the term “neutralizing agent” is meant any reagent (e.g., a solution,liquid, etc.) that when contacted with PCL, for example, brings the pHof the PCL to a neutral pH. Nonlimiting examples of neutralizing agentsinclude water and acidic solutions.

The terms “patient,” “subject” and “individual” are used interchangeablyherein, and mean a mammalian (e.g., human, rodent, non-human primates,canine, bovine, ovine, equine, feline, etc.) subject to obtain abiologic sample from.

The phrases “isolated” or biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany it as found in its native state.

The term “antibody” is meant to include polyclonal antibodies,monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies,anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled insoluble or bound form, as well as fragments, regions or derivativesthereof, provided by any known technique, such as, but not limited to,enzymatic cleavage, peptide synthesis or recombinant techniques.

The term “sample” is used herein in its broadest sense. A sampleincluding polynucleotides, polypeptides, peptides, antibodies and thelike may include a bodily fluid, a soluble fraction of a cellpreparation or media in which cells were grown, genomic DNA, RNA orcDNA, a cell, a tissue, skin, hair and the like. Examples of samplesinclude saliva, serum, blood, urine, buccal cells, and plasma.

Other features will become more apparent to persons having ordinaryskill in the art to which the package pertains and from the followingdescription and claims. Although devices, kits, and methods similar orequivalent to those described herein can be used in the practice ortesting of the present invention, suitable devices, kits, and methodsare described below. All publications, patent applications, and patentsmentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions, willcontrol. The particular embodiments discussed below are illustrativeonly and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects, features and advantages of the presentembodiments will be more apparent from the following more particulardescription thereof, presented in conjunction with the followingfigures.

FIG. 1 is a perspective view of a biologic sample collection device asdescribed herein in a biological sample extraction system.

FIG. 2 is a plan view of a biologic sample collection device kit asdescribed herein.

FIG. 3 is a schematic of one example of a method to produce the biologicsample collection device kit of FIG. 2.

FIGS. 4-8 are perspective views of several embodiments of a biologicsample collection device as described herein.

FIG. 9 is a perspective view of a mold for producing a biologic samplecollection device as described herein.

FIG. 10 is a micrograph of a PCL portion of a biologic sample collectiondevice described herein in a biological sample extraction system at 500μm.

FIG. 11 is a micrograph of the PCL portion shown inset in FIG. 10 at 200μm.

FIG. 12 is a micrograph of the PCL portion shown inset in FIG. 11 at 50μm.

FIG. 13 is a micrograph of the PCL portion shown inset in FIG. 12 at 10μm.

FIG. 14 is a graph comparing DNA yields using a biologic samplecollection device as described herein with controls.

FIGS. 15-21 are perspective views of several embodiments of a biologicsample collection device as described herein.

FIG. 22 is an exploded perspective view of one embodiment of a mold forproducing a biologic sample collection device as described herein.

FIG. 23 is an exploded perspective view of another embodiment of a moldfor producing a biologic sample collection device as described herein.

FIG. 24 is a perspective view of a biologic sample collection device asdescribed herein.

FIG. 25 is a perspective view of a biologic sample collection device asdescribed herein.

FIG. 26 shows one embodiment of a biologic sample collection device asdescribed herein. FIG. 26A is a schematic illustration of a biologicsample collection device, including holes that contain PCL and a label(e.g., an identifying label). FIG. 26B is a photograph of a biologicsample collection device having holes (apertures) in which PCL isdisposed or deposited.

FIG. 27 is a scanning electron micrograph (SEM) of an untreated PCLportion of a biologic sample collection device as described herein (leftpanel) and of a PCL portion of a biologic sample collection device asdescribed herein that was treated with NaOH (right panel).

Corresponding reference characters indicate corresponding componentsthroughout the several views of the drawings.

DETAILED DESCRIPTION

Despite advances in the art to improve the quality, cost and efficiencyof biological specimen collection devices, such as those made fromcotton, further improvements are desirable. Accordingly, one or more ofthe embodiments presented herein provide collection devices (and methodsof production and use thereof) related to biological sample collectionand more particularly, biologic sample collection devices and methodsrelated to DNA sample collection involving a biologic sample collectiondevice having a hydrophilic swab matrix that includes a syntheticcomponent. In a typical embodiment, the swab includes a modified PCL asthe synthetic component. The biologic sample collection devices, kitsand methods described herein are used to collect a biologic sample(e.g., blood, buccal cells, etc.) and to enable extraction of nucleicacids (e.g., DNA) from that biologic sample so that the nucleic acidscan be analyzed (e.g., sequencing and subsequent analyses of DNA).Generally, as referred to herein, “sample release” is the separationfrom or release of a biologic sample from a biologic sample collectiondevice as described herein. Specifically, “sample release” usuallyrefers to separation from or release of the biologic sample from thesoluble and hydrophilic PCL component of the biologic sample collectiondevice.

The following description is not to be taken in a limiting sense, but ismade merely for the purpose of describing the general principles ofexemplary embodiments. The scope of the embodiments should be determinedwith reference to the claims. Such biologic sample (e.g., biologicalspecimen) collection devices and methods can be useful in biomedicalapplications and nucleic acid (e.g., DNA) forensics.

Reference throughout this specification to “one embodiment,” “anembodiment,” or similar language means that a particular feature,structure, or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention. Thus,appearances of the phrases “in one embodiment,” “in an embodiment,” andsimilar language throughout this specification may, but do notnecessarily, all refer to the same embodiment.

To increase efficiency of sample release and to assure high quality ofthe nucleic acids (e.g., DNA) obtained, the present embodiments providealternatives to cotton as a swab material. The present embodimentsprovide materials that absorb biological samples efficiently, yet aresoluble in extraction reagents (e.g., extraction buffers). This resultsin high yields of DNA suited to DNA profiling using, for example, shorttandem repeats (STRs). In the embodiments described herein, syntheticmaterials in various combinations show superiority to cotton in theamount of DNA from control blood samples obtained using standardcommercially available DNA extraction kits. Using such kits, optimalcompatibilities between the biologic sample collection devices describedherein (at times referred to herein as “swabs”) and the extractionprocess can be determined. In some embodiments, the synthetic swabmaterials dissolve completely in an extraction buffer. A variety of swabcompositions for forensic applications can be designed, developed,optimized and validated.

Accordingly, in one embodiment, an element in the maximization of DNAyield can be a swab head that dissolves in DNA extraction medium, thuspreventing retention of absorbed sample by the swab head. In anotherapproach, improved tracking for a biological sample collection device isprovided by use of at least one unique identifier device including anRFID tag. Such a device can be situated in and/or on the biologic samplecollection device itself, on or in the packaging thereof, on a containerthereof (e.g., a specimen collection tube), and or on the cap of acontainer for a biologic sample collection device as described herein.These devices are paired with software that can store a variety ofdetailed information concerning the nature of the sample taken. Inanother approach, a kit is provided for use in forensic analyses thatcan perform the initial steps subsequent to sample collection (such assample preparation and/or analysis) and also to signal a user thatsufficient DNA has been obtained to allow complete analysis of thesample.

The biologic sample collection devices described herein are made frommaterials that do not react with a collected sample or specimen inunexpected ways, unless configured to do so, and preferably are noteffected by exposure to altered levels of various environmentalconditions, such as elevated ultraviolet (UV) light. In a typicalembodiment, sample/specimen profile analysis data is reportable in aformat usable with the Combined DNA Index System (CODIS). CODIS is anFBI funded database that stores DNA profiles created by US federal,state, and local crime laboratories. CODIS provides a searchabledatabase of DNA profiles to assist in the identification of suspects incrimes. The biologic sample collection devices as described herein areable to be manufactured in a scalable fashion (e.g., large scale) toprovide consistent and reliable swabs.

An additional challenge to biologic sample collection applications isthe assurance that the collected sample will yield sufficient DNA forsubsequent analyses (e.g., typing procedures needed to establish theidentity of the individual who was the sample source). Usingconventional techniques, this assurance is not available until the DNAfrom the specimen is amplified and the product is tested against knownquantities of DNA supplied as controls in the amplification kits. Thepresent embodiments can provide a reagent set that yields information onthe quantity of DNA obtained at the collection site. This allowscollection of additional samples should the amount be found inadequate.In one embodiment, a biologic sample collection device kit is providedthat allows initial steps of the analytic process to begin in the fieldand to give an indication of the quantity of DNA obtained. Such a kitcan include a biologic sample collection device packaged with DNAextraction reagents in lyophilized form. In this embodiment, a rapidamplification of DNA follows subsequent to extraction. Following theamplification of the extracted DNA, a colorimetric indicator can be usedas a signal that there is (or is not) adequate DNA for completeanalysis. This colorimetric indicator can be a DNA-indicating dyeincluded in the reagent, or alternatively, on an impregnated paper towhich a drop of solution is added. Methods involving conventionalmolecular biology techniques are described herein. Such techniques aregenerally known in the art and are described in detail in methodologytreatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology,ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York,1992 (with periodic updates).

Turning now to the figures, there is shown generally in FIG. 1 abiologic sample collection device as described herein positioned in abiological sample extraction system. Shown in FIG. 2 is one embodimentof a biologic sample collection device kit. FIG. 1 generally shows abiologic sample collection device 20 having a soluble and hydrophilicPCL 24 attached to a carrier 22. As shown in FIG. 1, DNA 30 from solubleand hydrophilic PCL 24 can be extracted while in an extraction solution28 (such as are commercially available to allow DNA profiling) disposedin an extraction system container 26. FIG. 2 illustrates one embodimentof a biologic sample collection device kit generally indicated at 31. Inthis embodiment, biologic sample collection device 20 is contained ordisposed within a first container 38, and the biologic sample collectiondevice includes a cap 32 which can be operably attached to the firstcontainer 38 such that the carrier and soluble, hydrophilic PCL 24 aresealed within the first container 38. In FIG. 2, a second container 34envelops and contains the biologic sample collection device 20 and thefirst container 38. Kit 31 can be sealed in a hermetically sealed secondcontainer (e.g., film package) 34 made from, for example, a polymerlaminate. As shown, second container 34 can be sealed at its ends 42,though other approaches, such as a three or even four sided seal arepossible. Second container 34 can have label 36 having identifyingindicia, which may include a bar code, an RFID tag 40, or both a barcode and an RFID tag. It is noted though that RFID tag 40 could also bepositioned on carrier 22 of collection device 20 or first container 38.Also, as shown in the figures, RFID tag 40 can be positioned on top ofthe cap (e.g., seal) 32 (FIG. 24) or within the cap (e.g., seal) 32(FIG. 25). In these last two embodiments, the identifier and the sampleare not separable prior to analysis. A biologic sample collection deviceor kit as described herein can include one or more of: an RFID tag, abar code, and a label (e.g., two or more of an RFID tag, a bar code, anda label; all of an RFID tag, a bar code, and a label).

The importance of specimen identification, whether by indicia, RFIDtags, bar codes, and the like, and combinations thereof, cannot beunderstated. Sample collection devices, such as in forensicapplications, are used to obtain samples of biological materials forsubsequent analyses that serve to establish the identity of the samplesource by subsequent analytic steps. The sample collection device may besubject to analyses immediately or it may be stored for some period oftime prior to analysis. Often multiple samples must be taken (forexample at a crime scene). In this instance the samples may not beanalyzed for long periods of time because of a backlog in thelaboratory, or the need to send samples for tests not available near thescene of collection. Therefore, it is important that certainidentification criteria accompany the sample. Current procedures in theart for providing a clear and secure means to locate and identify thesample at any stage in the process depend mainly on a bar coded identityof the sample collected on the device and handwritten information onpackets in which samples are placed upon collection. The presentembodiments provide improvements to the accuracy, quality and quantityof sample information handling.

In addition to the standard barcode or quick response (“QR”) codecarrying a manufacturer's information and identifier, as is known in theart, the present embodiments provide a passive RFID label (an RFID tag)used to tag the device. The RFID tag can be imprinted with information,such as a bar or QR code. This manufacturer-supplied information caninclude lot and serial number to unambiguously identify the collectiondevice. In one embodiment, the RFID tag can be further programmed toinclude user-supplied data that relates to the sample taken in aspecific manner. This provides an additional advantage over bar codesthat use a simple scanning device to allow users to scan collections ofsamples in a storage area, such as a refrigerator or freezer. Here, useof a programmable RFID tag allows obtaining of information (both userand manufacturer supplied) on the tag without removing and scanning eachunit individually as with samples having only bar or QR codes.

Most instruments used for analyses of the samples retrieved from abiologic sample collection device described herein can also beprogrammed to read the tag and thus incorporate all information alongwith the results for easy and secure display. The inseparability of thesample and its detailed identity and the ability to locate specificsamples with relative ease can greatly aid forensics applications where,for example, a chain-of-custody needs to be documented to be admittedinto evidence in court proceedings. An example of how a programmableRFID tag can be implemented in forensic specimen collection can beinitiated as follows. Upon completion of manufacture, a given lot orbatch of biologic sample collection devices or kits as described herein(e.g., biologic sample collection swabs) is assigned a lot number whichincludes an identity code of the product (for example P120302) and eachunit is given a serial number such as 1001 to 9999. These identifiersalong with additional company-supplied data are programmed into the RFIDtag and additionally imprinted on a bar or QR code printed on the tag.These labels are printed at the time of manufacture using a commerciallyavailable printer.

When a user collects a sample with a biologic sample collection deviceas described herein, in one approach they may add additional informationof their own choosing to the RFID tag by use of a portable programminginstrument. This user-supplied data may include, but is not limited to:the circumstances for taking the sample (offense, victim, suspect), theplace and time at which the sample was taken, the source of the sample,the identity of the person(s) taking the samples, and the like. Thesedata can be used to establish the authenticity and origin of the samplesand, with a clear record of the chain-of-custody for the sample,represent information for the sample to be admitted into evidence incourt proceedings. Using conventional methods, sample/specimenidentification is either by written record accompanying the samplecollection device or by a barcode present on the container in which thedevice is stored awaiting analysis. Unless the identifying barcode isinseparable from the collector, problems may occur from separation ofsample from this identifier. Typically, the bar or QR coded informationyielded upon scanning cannot be expanded after its initial printing sono user-specific information is included.

In a typical embodiment, once entered, no data on the RFID tag may beerased or altered. It is noted that as technology improves, the amountof information present on the RFID tag will increase. At present, acommercially available and economical RFID tag can have themanufacturer's identifier and an additional 60-70 characters from theuser. RFID tags now in the testing phase have 10 times that capacity.Use of RFID tags is known in the art and is described, for example, inU.S. Pat. No. 7,978,074.

The exact location of the tag on the collection device may vary and thesignal from the tag is preferably readable within at least 5 feet of thetag. If the tag is in a visible place the bar or QR code can also bescanned to provide further verification of the identity of the device orkit. To enhance reliability of the RFID tag, it can be attached to abiologic sample collection device or kit with an adhesive that rendersthe tag useless if accidental or intentional removal is attempted.

Those skilled in the relevant art will appreciate that the embodimentsdescribed herein can be practiced with any of various communications,data processing, or computer system devices, including: hand-helddevices (including personal digital assistants (PDAs)), wearablecomputers, cellular or mobile phones, multi-processor systems,microprocessor-based or programmable consumer electronics, network PCs,mini-computers, mainframe computers, and the like. Aspects of theinvention described herein may be stored or distributed oncomputer-readable media, including magnetically or optically readablecomputer discs, hard-wired or preprogrammed chips (e.g., EEPROMsemiconductor chips), nanotechnology memory, biological memory, or otherdata storage media. Computer-implemented instructions, data structures,screen displays, and other data under aspects of the invention describedherein may be distributed over the Internet or over other networks(including wireless networks), on a propagated signal on a propagationmedium (e.g., an electromagnetic wave(s), a sound wave, etc.) over aperiod of time, or they may be provided on any analog or digital network(packet switched, circuit switched, or other scheme).

First container 38 can be made from a variety of materials such as aglass tube that is closed at one end and configured to receive abiologic sample collection device that includes a cap (e.g., seal) 32 ata second end. The seal formed between cap (e.g., seal) 32 and firstcontainer 38 preferably provides a watertight seal in up to twoatmospheres of pressure at ambient temperatures (e.g., about 20-25degrees Celsius). Cap 32 can be made of any suitable material, e.g.,rubber, plastic, or other synthetic polymer. First container 38 can alsoinclude an identifying label 44. Cap 32 can be connected to carrier 22using a variety of means such as by a non-reactive glue, a pressure fitinto a seal bore (not shown), and the like. The bore to receive thecarrier 22 can be a variety of shapes designed to retain a carrier endhaving a reciprocal shape. In one embodiment of an assembled biologicsample collection device as described herein, the soluble, hydrophilicPCL portion 24 has at least about a 1 mm clearance from the sides offirst container 38, but can preferably range from about 1 to 4 mm (e.g.,0.8 mm to 5.0 mm). The soluble, hydrophilic PCL portion 24 is typicallyalso disposed within the first container 38 such that it has a suitableamount of clearance from the tip or bottom (e.g., portion or endopposite the cap 32) of the first container 38, e.g., at least about a 1mm (e.g., 0.8 mm, 1.0 mm, 1.1 mm, 1.5 mm, etc.) clearance.

FIGS. 4-8 and 15-21 show non-limiting examples of various shapes andconfigurations of the soluble, hydrophilic PCL portion 24 of a biologicsample collection device 20. A nonlimiting list of shapes includes:cubes, cuboids, cones, sphere, cylinders, spheroids, prisms,square-based pyramids, triangular-based pyramids, essentiallytwo-dimensional shapes and configurations, etc. Variables affectingshape can include consideration of the sample origin to be collected,manufacturing efficiency, material economy, storage, clearance withinfirst container 38, and the like and combinations thereof. It is notedthat even a flat sheet of soluble, hydrophilic PCL 24 as shown in FIG.21 can be used, e.g., used in filter paper tests in mandated new bornscreening programs. Also, FIG. 7 shows an optional band 46 to retainsoluble, hydrophilic PCL. FIG. 8 shows an additional collar of soluble,hydrophilic PCL 48. It is noted that this type of collar 48 may be theresult of overfill during a molding process described herein.

Materials selected for use in a biologic sample collection devicepreferably provide no or limited adverse affect to targeted biospecimensamples and are easy to handle or use. Such materials (e.g., soluble,hydrophilic PCL) efficiently adsorb samples of various types such asbodily fluids, cells shed from fingerprints, and the like. Suchmaterials should release samples with high efficiency using, forexample, commercially available DNA extraction kits. Typically, thematerial (e.g., modified PCL) added to or disposed on or within thecarrier is soluble with no unintended interference with subsequent testsand analyses. Such solubility reduces sample handling and provides agreat advantage of being able to go immediately into robotic analyticsystems. The biologic sample collection devices described herein have areasonable cost of manufacturing, yet provide product consistency. PCLis strong, but soft and foamy. It is an easily manufactured andbiodegradable polyester with a low melting point of around 60° C.However, PCL has low hydrophilicity (i.e., a low affinity for water; noteasily absorbing or dissolving in water). As described in more detailbelow, the biologic sample collection devices include PCL that has beenmodified or treated to be soluble and hydrophilic.

PCL is a monopolymer made by a ring-opening polymerization of epsiloncaprolactone. Similar polymers are polylactide, polyglycolide orpolydioxanone. PCL may be copolymerized with other esters such aspolylactide to alter properties. Polymers of acrylamide may also beused, such as poly N-isopropylacrylamide. The addition of derivativegroups to the PCL polymerization reaction may be used to changeproperties of the PCL. For example, the carbohydrate trehalose can beused to enhance DNA stability.

Stability of collected biological samples is critical for accurateanalysis and profiling. Results obtained from analyses of the collectedsamples should be comparable irrespective of the time interval betweencollection and analysis. Typically, analyses are not performedimmediately after sample harvest and there are a number of chemical andbiological agents and conditions that can affect the integrity of thesample or of the DNA from it. Insulation from atmospheric conditions bystorage in temperature and humidity controlled areas is common topreserve sample integrity. Several methods are known to preservepurified DNA from degradation after primary processing of the samples atthe analytical laboratories. As indicated above, a carbohydrate,trehalose, is one of the compounds currently used as a stabilizer fordried DNA during storage. It is most effective when the purified DNA isstored at about of range of −60° C. to −90° C., and preferably about−80° C. or at ambient temperature (e.g., about 18° C. to 25° C.). Apossible mechanism of action is that trehalose replaces water moleculeslost during desiccation. In one embodiment, trehalose can be mixed withPCL or other swab materials to increase the hydrophilicity of the PCLand, at the same time, give greater stability to the DNA in the samplefrom the moment of collection.

In another approach soluble, hydrophilic PCL 24 can be impregnated witha bacteriostatic or fungicidal substance to inhibit bacterial growth forsamples in storage. Other possible modifications include inhibitors ofenzymes (such as DNAse or other nucleolytic enzymes) that can degradethe sample. Such modifications are amenable to any of the consideredswab material formulations. In other approaches, soluble, hydrophilicPCL 24 can be modified by coupling a protein to PCL, such as anantibody.

PCL as a swab component is unknown since it typically is not suitable tocollect biological samples since hydrophilicity is an important elementfor a collection swab composition. Accordingly, the PCL as presentedherein, is modified to improve its hydrophilicity. Such PCL modificationpreferably enhances absorbency, sterility and freedom from contaminatingDNA. PCL can be modified using any suitable chemical or physicalmethods. One or more surface features of PCL can be modified (or added)to increase hydrophilicity.

Solutions used in the production of soluble, hydrophilic PCL 24 can besterilized using filtration where sterility is maintained by handlingthe assembly and packaging of the product in a clean room environment.Alternatively, a biologic sample collection device (e.g., a swab asdescribed herein) or specimen/sample collection kit can be sterilizedusing UV or gamma irradiation of the finished packaged product. Thisprocedure can also increase wettability and hydrophilicity of the PCLswab.

PCL for use in the devices, kits and methods described herein can bemixed with several chemicals during manufacture to give varyingproperties and a desired amount of hydrophilicity. For example, Ca(Cl)₂can be added during a solubilization step of the PCL. As anotherexample, PCL can also be treated with a solution of Ca(OH)₂ or NaOH.Both methods can be used to increase the hydrophilicity of the PCL. Itis noted though that soluble, hydrophilic PCL 24 can be coupled toand/or copolymerized with a variety of other materials in addition toPCL in various proportions and combinations to yield a biologic samplecollection device (e.g., a swab as described herein) with the desiredabsorbency and solubility. For example, the soluble, hydrophilic PCL caninclude alginate, silk or other natural fibers.

PCL can also be mixed with several proteins like silk, collagen, fibrin,antibodies, or the like, or various combinations thereof to increase thesame. Such proteins can be extracted from natural sources or producedusing recombinant organisms expressing the proteins of naturallyoccurring cloned genes or from synthetic genes. In one approach wherethe protein is silk, the silk is purified and ground to a fine powder,and then mixed at about 1 to 3 percent weight/volume of the PCLsolution. Where the protein is a genetically engineered protein it canhave defined properties and expressed in vitro.

Soluble, hydrophilic PCL 24 can also be coupled to a geneticallyengineered protein with defined properties and that can be expressed invitro. For illustrative purposes only, in one approach, genes can besynthesized coding for protein sequences containing the repeat found inBombix mori crystalline region of silk (GAGAGS) (SEQ ID NO:1) and thepentapeptide of human elastin (GVGVP) (SEQ ID NO:2) to form proteins ofabout 60 kDa. Through recombinant DNA technology procedures, theproteins can be expressed and purified from a variety of expressionorganisms including but not limited to bacterial strains such as E. colior B. subtilis, or higher organisms such as fungi, yeasts human celllines such as CHO, and the like. In the same manner, genes encodingvarious collagen sequences can be synthesized and purified. Theseproteins can be shaped into fibers, sponges, and the like.

Additionally, proteins incorporated into soluble, hydrophilic PCL 24 canbe designed to contain, interspersed at regular intervals, specificsequences to be utilized in chemical reaction or biological functions.As an example only, such sequences could contain the amino acid Lysine(to introduce an active primary amine) or binding site such as RGD (tobind specific cells).

In one format, the PCL swab may be configured for use in systems thatrequire small sample size in the extraction step of the analyticprocess. Typically in current usage, a sample is taken by buccal swabonto a paper collector. This collector is then punched to obtain a small(1.2 mm) sample of the paper containing the sample which is placed intothe extraction mixture directly. Because the buccal sample is relativelyrich in cells from which DNA may be extracted, the small sample sufficesto yield material for the analytic process. However, there is a failurerate to the process that may be due to an uneven distribution of cellson the paper. Taking a larger punch from the paper can interfere withdownstream steps of extraction and amplification.

A biologic sample collection device (e.g., biologic sample collectionswab) tailored for this purpose using PCL is described herein. In thisembodiment, the swab handle is plastic (or hard non-porous wood) in asemi-cylinder or flat ovoid shape with one or more holes, dimples,apertures, depressions, etc., into which PCL has been introduced. Thebiologic sample collection device is used as a collector of buccal cellswherein the PCL areas take up cells with greatest efficiency compared tothe hard wood or plastic surrounding material. The PCL contained in theholes is then pushed through the carrier (e.g., swab handle) into a tubecontaining extraction mix and the process of extraction, amplificationand analysis begins. The implement for removing the PCL plugs from thehandle resembles a toothpick and is sterile, DNA free and non absorbing.In this embodiment, the fact that the PCL contained in the hole isexposed on two sides of the carrier gives it good accessibility to cellsin the sample. Although exposure on two sides is unnecessary, it doesallow good access to cells in saliva and makes it easy to remove the PCLby punching it out into a tube, for example. The wetting of the PCL withsaliva that occurs in the buccal collection event will facilitate uptakeof material into the PCL which is the only absorbent material on thebiologic sample collection device. A further advantage is given by thefact that the PCL is soluble in most extraction reagents and does notinterfere with analyses allowing a sample larger than the 1.2 mm paperpunch to be used.

The PCL used for this purpose may be prepared in sheets and conditionedto be hydrophilic prior to coupling the PCL to a carrier (e.g.,introduction into the swab handle). Alternatively, it may be introducedin the liquid form and conditioned (treated or modified) while coupledto a carrier. Co-polymers of PCL and mixes with other materials may beused to maximize uptake of saliva and cells into the PCL portion(s) of abiologic sample collection device as described herein.

In the biologic sample collection device shown in FIG. 26, there are atleast two areas into which PCL is introduced. Such a biologic samplecollection device may be particularly useful for reference samplecollection. In some embodiments, the collected sample or specimen may betaken from only one of these while the other is stored for future use. Alabel is affixed to the sample with bar or QR code to allowidentification of the sample to remain with the material. FIG. 26 showsone embodiment of a biologic sample collection device for referencesample collection, which allows facile collection of cells such asbuccal cells, for example.

Turning now to FIG. 3, there is shown a schematic diagram of one methodto produce a biologic sample collection device kit as described herein.It is noted that at ambient temperatures (e.g., between about 20-25degrees Celsius, with an average of about 23 degrees Celsius) PCL issoluble in a variety of solvents including chloroform, toluene,2-butanone, dichloromethane, acetone, ethyl acetate, acetonitrile, andthe like and combinations thereof. Accordingly, several of thesesolvents can be used to form the biologic sample collection devices(e.g., biologic sample collection swabs) of the present embodiments, butfor illustrative purposes in the method described, PCL is solubilized inglacial acetic acid. Acetic acid is a concentrated vinegar, but it isotherwise a fairly weak acid and not regulated as a carcinogen andtherefore not regulated for safety handling. Preferably the pH range ofthe solvent is about 4-10. This range allows for easier disposal usedmaterials. In short, acetic acid in this pH range allows large scale andeconomical swab production.

Beginning at step 50, a solution is prepared having a matrix formingmaterial, such as PCL, between about 3 percent and 10 percent(preferably between about 4 percent to 7 percent and most preferably atabout 5 percent) weight/volume in the specified solvent, typicallyglacial acetic acid. Other components can include other co-polymers,silk fiber, synthesized proteins (recombinant proteins structural andfunctional) and the like. The other co-polymers can include PMMA, LPGA,and the like. Further, trehalose (a tri-saccharide) can also be acomponent of the solution as well as chitosan, and the like. It is notedthough that even as the composition component varies, the overallpercent weight/volume as stated above for the matrix forming material ispreferably mantained.

Next, at step 52, the solution is incubated. The PCL solution can bedelivered to a bath kept at about 60° C. to 70° C. In one approach, thetemperature chosen can be about 65° C. with optional stirring, such asfor about 2 hours, for usually about 1 to 4 hours of incubation. At step52, if a solvent other than glacial acetic acid is used, the incubationcan be maintained at or near ambient temperature. It is noted that theincubation can occur at room temperature for any bath, but the increasedtemperature is used to reduce incubation time. At ambient temperature,incubation could take about 24 hours. Stirring also creates homogeneousmixture, so it does not need to be stirred continuously, if at all.Nevertheless, stirring can reduce incubation time and increaseshomogenicity.

Once incubated, the process can move to step 54, where the PCL solutionis cooled at ambient temperature, if needed. As volume increases, it mayneed to be cooled under temperature control. A heat exchanger can beused to bring temperature to ambient. Stirring could also be used in atemperature controlled environment. In one approach, the incubatedsolution is allowed to equilibrate to an ambient temperature, such asbetween about 20-25° C. This step facilitates the handling of thesolution and reduces evaporation rate of the solvent thus reducingchange in the desired concentration of PCL. This temperature can bedifferent based on the characteristics of the solvent used to generatethe PCL solution. This incubation and cooling step is preferably in anenclosed environment so as to minimize changes in percent volumes of thesolute.

Next, at step 56, the PCL solution equilibrated to ambient temperatureis delivered to molds having wells that are configured to be in theshape of the desired swab, such as the exemplary swabs shown in FIGS.4-8 and 15-21. Several forms can be used depending on the final use ofthe swab or even as a sheet of material (FIG. 21). Exemplary moldconfigurations are shown in FIGS. 9 and 22-23. Molds can be formed frommetals and the like and even coated with a fluoropolymer coating such ason sold under the trade name TEFLON®. Mold materials should be selectedto have no or little reaction with the solute. For example aluminum iscorrosive in the presence of an acid. As shown, molds 74 can be formedas opposing halves that can be clamped together by a clamping deviceextending through holes 76 and can provide a plurality of wells 80. Asshown in FIG. 23, molds can be clamped together in a series and orientedwith precision using pins 82 extending into reciprocal holes (not shown)on the adjacent mold. Here wells 80 are on both sides of the mold half.

Next, at step 58 stems (i.e., carriers) are placed where desired throughmold well 80 to allow the solution to envelop the swab and form a swabassembly. Stems may have, at one end, a cap/seal that fit the tube(i.e., a first container) used in step 70 as described above.

Once stabilized (i.e., stems are in place and the solution with matrixmaterial is in the mold well 80), at step 60 the swab assembly is cooledbelow ambient temperature. In one approach, the assembly is exposed toan environment of about −20° C. (range of about 18-30 degrees Celsius).This step can last from 4 to 24 hours, usually it lasts 6 to 18 hrs. Afurther cooling step can also optionally be applied to swab. Forexample, in this additional step the environment is maintained at about−80° C. for about an additional 2-8 hours, most frequently for about 4to 6 hours. The rate of cooling should be at a rate that will not causecrystallization. Accordingly, any exposure to about −80 degrees Celsiusmay not provide an acceptable product.

Following the cooling step, the assembly can be removed from the mold atstep 62 and submerged in liquid nitrogen to avoid melting if there isany delay before going to step 64. This step maintains the temperatureuntil the lyophilization step below.

Next, at step 64 the swab assembly is freeze dried (lyophilized). Thisstep can last for about 20-24 hours in a chamber of about −50 degreesCelsius (−40 to −60 range). Alternatively the drying step can be done atambient temperature for 2 to 4 days depending on the solvent used andthe shape and size of the swab. Ambient temperature drying may be mostviable in sheet embodiments (FIG. 21).

Following lyophilization or drying, the assembly can by treated (washed)at step 66 with a strong base solution (e.g., having a pH greater thanabout 12) and tested for water uptake at step 68. Such treatment can useabout 2 N NaOH (sodium hydroxide), and then the swab is neutralized,treated, and dried. This step can increase the swab's ability to take upwater (i.e., its hydrophilicity). The NaOH treating step may partiallyhydrolyze the swab to expose hydroxyl groups. Potassium hydroxide andother types of bases may also work. This treatment step may also improvehydrophilicity with swabs made from other copolymers. Optionally, othertypes of hydrophilic proteins can be used for the swab matrix thateliminate the need for this step. PCL is still preferably used becauseit works best with DNA extractions. The yield of DNA is not only good,but it also can allow for better or more amplification. Prior art cottonswabs do not release DNA as well as shown in FIG. 14, which is describedbelow. In one embodiment, referring to step 68 of FIG. 3, swabs areimmersed in a beaker with 2 N NaOH and placed on a shaker for 24-48hours at room temperature. They are then treated until the treatmentwater has a neutral pH (e.g., a pH of about 7). Usually 5 treatments aresufficient. The swabs as described herein can then be allowed to dry for18-24 hrs either in an open container or under minimum vaccuum. Atpredetermined production intervals the resultant swabs can be tested byexposure to 50 μl of water containing 0.2 percent trypan blue. Previousto treatment, the solution forms a bead on the surface of a PCL swab.After successful treatment of the PCL swab as described above, thecolored water spreads over the surface of the swab which absorbs asignificant fraction of the 50 μl. A further test involves weighing ofthe swab before and after exposure to 50 μl of water; a weight increaseof at least 40 mg (40 μl) indicates that the swab is sufficientlyhydrophilic for use.

It is noted that long exposure to NaOH (e.g., more than 3 days) orexposure at elevated temperatures (e.g., greater than about 37 degreesCelsius) will result in dissolution of swab head. Similarly, exposure tostrong acids has no effect on the hydrophilic properties of the PCL.

Next, at step 70 the swab assembly is finalized by combining the swabwith tubes, sealing caps, packaging, and the like. Assembly ispreferably done in a laminar flow hood. Clean rooms and sterileenvironments can also be beneficial.

Next, at step 72, the swab assembly can be sterilized. In one approachthis can be by UV light exposure, though other methods, such as gammairradiation, are possible so long as the treated swab matrix integrityand desired water uptake is maintained. This step sterilizes and rendersthe swab DNA free. In one approach, UV exposure can by about 20 minuteswith 5000 microjoules/cm². In another approach, UV radiation can be intwo 15 minute cycles of 6000 microjules/cm² using exposure indicators inthe chamber and in control packages to assure exposure. Biologic samplescollected for use in identification of individuals should providerigorous extraction and amplification to obtain a quantity of DNA ofsufficient quality for use in assays to determine allelic forms ofmultiple pre-selected genes, such as used for developing DNA profiles.Conventional collection swabs have characteristics that render themunusable for the required analyses when paired with certain commonlyused commercial DNA extraction kits. In some embodiments, a swab asdescribed herein is soluble in DNA extraction solutions of detergentshaving, for example, protease K. In other embodiments, the swab issoluble in DNA extraction solutions having a chaopropic agent.

A swab as described herein can be composed entirely from modified(treated) PCL or derivatives or copolymers thereof. For example, in someembodiments, one can use PCL or a PLC derivative material to surfacemodify a known preformed swab composition. For example, a commerciallyavailable polyurethane foam swab used for a variety of purposes (e.g.,cleaning, applying paint, make-up etc) can be dipped in PCL to providethe advantages of PCL with special shapes. Such embodiments provide achoice from the large selection of existing swabs and thus selectingthose of optimum shapes for given applications. The PCL used in this waycan be prepared and applied to the existing swab (substrate) in severalways, which is facilitated by the adherence of PCL to hydrophobicsubstances.

Although a typical embodiment of a biologic sample collection deviceincludes soluble and hydrophilic PCL coupled to a carrier, in someembodiments, no carrier is required. For example, one could use a pieceof soluble, hydrophilic PCL (e.g., obtained from a sheet of soluble andhydrophilic PCL or removed from a biologic sample collection device asdescribed herein and manipulate it with a forceps) for collecting abiologic sample from which nucleic acids can be extracted.

EXAMPLES

The present invention is further illustrated by the following specificexamples. The examples are provided for illustration only and should notbe construed as limiting the scope of the invention in any way.

Example 1 Treatment and Modifications of PCL

FIG. 27 shows scanning electron micrographs of a PCL swab that wastreated with NaOH in the following manner. The swab was treated with 2 NNaOH for 18 hrs at room temperature, neutralized by washing with wateruntil washes were below pH8 and dried. The swab treated in this mannerwas compared (at the same magnification) to one prior to such treatment.Note that the untreated swab is smoother and lacks the fuzzy surface ofthe NaOH treated swab.

FIGS. 10-13 show photomicrographs of the surface of a modified PCL swabat 500, 200, 50 and 10 μm. These micrographs show that the treated PCLhas a deformed and crumpled appearance having even pores betweenpatches.

The modified PLC swab materials prepared as described herein were testedfor DNA yields, the results of which are shown in Table 1 below and FIG.14.

TABLE 1 Extraction Total DNA Std Fraction of blood alone Description Kityield (ng) Dev (×100 = %) PCL Swab Kit A 57.9 19.1 0.71481481 Kit B 1058.2 0.91304348 Kit C 49 12 0.76323988 Control Kit A 37.5 14.8 0.46296296Swab 1 (cotton) Kit B 63.8 10 0.55478261 Kit C 10.6 1.4 0.16510903Control Kit A 58 16.5 0.71604938 Swab 2 (Poly- Nipaam) Kit B 25 17.70.2173913 Kit C 0 0 0 Blood alone Kit A 81 4.2 1 Kit B 115 21.2 1 Kit C64.2 26.5 1

As shown, the modified PCL swabs were tested with a variety of commonlyused, commercially available DNA extraction and amplification kits(Labeled Kit A, B and C). Comparisons of the three kits with modifiedPCL swab, standard cotton (control swab 1) and a swab made from variousengineered materials (e.g., control swab 2 of Poly-Nipaam) show higherDNA yields from the controls.

It was also observed that under defined conditions, the head of themodified PCL swab dissolves in the DNA extraction reagents and the smallamount of solute remaining is removed by an early process step. ThisDNA-containing solution may then be used to carry out subsequentanalytic steps with no or minimal interference from the dissolved swabmaterial. The extracted DNA from the swabs of the present embodiments isbiocompatible with PCR and STR typing procedures.

Specifically, as shown in FIG. 14, results for 3 different commonly usedDNA extraction kits with a modified PCL swab compared to one made withpoly N isopropylacrylamide (Nipaam) and to one made with cotton areshown. While improved performance was seen with the treated PCL swabcompared to cotton using Kit A (about 45 versus 65 percent), a muchgreater improvement was seen with Kit B (about 40 versus 90 percentrecovery). In terms of absolute yield the Kit B was best but the resultswith Kit C showed the greatest improvement for PCL versus cotton (about17-62% or almost 4 fold increase). This result suggests that pairingswab variations with identified DNA extraction process can yield bestresults. It was noted that the PCL swab was soluble in the extractionbuffers of all kits tested, whereas control swab 2 formed a slurry orwas otherwise insoluble.

Example 2 Production of a Modified PCL Preformed Swab

One example of a method to produce a modified PCL preformed swab isprovided as follows. Commercially available PCL pellets can be dissolvedin an agent, such as glacial acetic acid or dichloromethane, or glacialacetic acid and dichloromethane in combination at concentrations fromabout 2 percent to about 10 percent weight/volume, preferably about 3.5percent to about 7 percent weight/volume, and most preferably about 5percent. It is noted that variation of concentrations produces a coatingof different pore size. Preformed swabs can be dipped into the compositesolution for 5 to 10 minutes to form a uniform coating. The coated swabsare air dried, for example for up to 24 hours. Alternately, PCL coatedswabs can be frozen and freeze-dried in a manner described herein forPCL swabs to give desired texture of the PCL. One can also coat theexisting swabs with a solution of PCL mixed with a salt, such as sodiumchloride in a solution of about equal parts of PCL and the salt. Next,the dried PCL coated swab can be submerged in an aqueous solution toallow the salt to leach out of the swab. Porosity of the finished swabcan be varied by altering volume percentages of sodium chloride in thepolymer-solvent slurry. Coating thickness can be controlled by thenumber and duration of immersions in the dip-coating technique. In thismethod, the coating can be accomplished by short (e.g., five seconds toone minute) successive dipping (e.g., once, twice, three times, fourtimes, etc.) of the swab into the PCL solution. The dipped substrate canbe air dried for about 10 to 15 minutes between dipping cycles to applymore uniformly the PCL to the substrate. The treated swab can then beair dried.

As a further variation, a caprolactone monomer can be copolymerized withother monomers such as lactic acid, PEG, and the like. Theseco-polymerizations can be used to change the properties and behavior ofthe material including its porosity and hydrophilicity.

Example 3 Pick-Up and Release of DNA from Human Blood Samples

In Table 2, “pick-up and release” data is shown. Pick-up and release ismeasured by swabbing a glass slide to which was added 1 ul of humanblood, and extracting and quantifying DNA from the swab. Release ismeasured by dropping the blood sample directly onto the swab, allowingit to dry and quantifying DNA as above. A control for both is adding theblood directly to the extraction tube. The data in Table 2 reveal thatPCL swabs were an average of 55% efficient when used to pick up a samplefrom a glass slide and then release the DNA from that sample in acommercial extraction buffer. This number measures pick-up and releaseof samples. In order to measure release of DNA from sample, the bloodsample was dropped onto the swab directly, allowed to dry and the DNAwas then extracted. In this case 68% of the DNA in the sample wasobtained. Both of these experiments use the amount of DNA from bloodssample dropped directly into the extraction buffer as 100% value.

TABLE 2 Pick-up and release of DNA from human blood samples: Sample NgDNA % control Pick-up & release 12.5 55 Release 15.7 68 Control 23 100

In Table 3, measurements of pick-up and release from swabs retained forup to 7 days at ambient temperature were obtained. The data listed inthis table indicate that swabs may be retained after acquisition of ablood sample for at least 7 days with no significant loss of DNA fromthe sample when compared to a swab that is used immediately after sampleacquisition.

TABLE 3 Aging of Blood Samples on X-swab (a biologic sample collectiondevice as described herein) DNA obtained Day (ng)** % of control* 0 12.255 3 12.9 58 5 13.2 59 7 11.5 52 *Control consists of 1 ul of humanblood added directly to DNA extraction tube; average yield was 22.2 ngDNA **Results are average of 4 determinations in which 1 ul dried bloodwas acquired from a glass slide and X-swab extracted using Qiagenreagents on the day after sample collection indicated.

Example 4 Production of Swabs or Swab Materials Using NaOH or NaHCO₃

In one example of a method of producing a biologic sample collectiondevice as described herein, the method involves use of NaOH. Oneembodiment of such a method includes the following steps: dissolving PCLin a PCL dissolving agent at a concentration in the range of about 2percent to about 10 percent weight/volume, resulting in a PCL compositesolution; submersing a carrier at least once into the PCL compositesolution such that the PCL composite solution coats the carrier; dryingthe PCL composite solution-coated carrier; submersing the PCL compositesolution-coated carrier in an aqueous solution, whereby the dissolvingagent is leached from the PCL composite, resulting in a PCL-coupledcarrier; contacting the PCL with a base having a pH greater than 8(NaOH); washing the PCL with water, or acidic solution, until the PCLhas a neutral pH; drying the PCL; and sterilizing the PCL-coupledcarrier and rendering it free of nucleic acids, resulting in a biologicsample collection device comprising soluble and hydrophilic PCL coupledto the carrier, wherein the PCL solubilizes when exposed to a nucleicacid extraction reagent.

In another example of a method of producing a biologic sample collectiondevice as described herein, the method involves use of NaHCO₃. Oneembodiment of such a method includes the following steps: dissolving PCLin a PCL dissolving agent at a concentration in the range of about 2percent to about 10 percent weight/volume, resulting in a PCL compositesolution; submersing a carrier at least once into the PCL compositesolution such that the PCL composite solution coats the carrier;submersing the PCL composite solution-coated carrier in an aqueoussolution having a pH greater than 8 (NaHCO₃), whereby the dissolvingagent is leached from the PCL composite, resulting in a PCL-coupledcarrier; washing the PCL with water, or acidic solution, until the PCLhas a neutral pH; drying the PCL; and sterilizing the PCL-coupledcarrier and rendering it free of nucleic acids, resulting in a biologicsample collection device comprising soluble and hydrophilic PCL coupledto the carrier, wherein the PCL solubilizes when exposed to a nucleicacid extraction reagent.

Other Embodiments

Any improvement may be made in part or all of the devices, kits andmethod steps. All references, including publications, patentapplications, and patents, cited herein are hereby incorporated byreference. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended to illuminate the invention anddoes not pose a limitation on the scope of the invention unlessotherwise claimed. Any statement herein as to the nature or benefits ofthe invention or of the preferred embodiments is not intended to belimiting, and the appended claims should not be deemed to be limited bysuch statements. More generally, no language in the specification shouldbe construed as indicating any non-claimed element as being essential tothe practice of the invention. This invention includes all modificationsand equivalents of the subject matter recited in the claims appendedhereto as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contraindicated by context.

What is claimed is:
 1. A biologic sample collection device consistingof: soluble and hydrophilic polycaprolactone coupled to a carrier,wherein the polycaprolactone is optionally coupled to one or more of acarbohydrate trehalose derivative group and at least one protein, andthe carrier optionally comprises one or more of an identifying label, aradio-frequency identification tag and a bar code, and wherein at leasta portion of the polycaprolactone solubilizes when exposed to a nucleicacid extraction reagent.
 2. The biologic sample collection device ofclaim 1, wherein the polycaprolactone is copolymerized with at least oneagent selected from the group consisting of: an acrylamide and apolyester other than polycaprolactone.
 3. The biologic sample collectiondevice of claim 2, wherein the at least one agent is selected from thegroup consisting of: polylactide, polyglycolide, polydioxanone,acrylamide, poly N-isopropylacrylamide, and polyurethane.
 4. Thebiologic sample collection device of claim 1, wherein the biologicsample collection device has been sterilized.
 5. The biologic samplecollection device of claim 1, wherein the polycaprolactone has beentreated with a base having a pH greater than 8 and a neutralizing agentfor increasing hydrophilicity.
 6. The biologic sample collection deviceof claim 5, wherein the base is selected from the group consisting of:NaOH, NaHCO₃, KOH, Na₂CO₃, and CA(OH)₂.
 7. The biologic samplecollection device of claim 1, wherein the at least one protein isselected from the group consisting of: antibody, silk, collagen, fibrinand elastin.
 8. The biologic sample collection device of claim 1,wherein the carrier comprises a material selected from the groupconsisting of: metal, plastic, polymer, wood, and glass.
 9. The biologicsample collection device of claim 1, wherein the biologic sample is ahuman buccal sample.
 10. The biologic sample collection device of claim1, wherein the polycaprolactone is contained within at least oneaperture disposed in the carrier.
 11. The biologic sample collectiondevice of claim 10, wherein the polycaprolactone is contained within twoor more apertures disposed in the carrier.
 12. The biologic samplecollection device of claim 10, wherein the at least one aperture isdisposed at a first end of the carrier, and an identifying label isdisposed at a second end of the carrier.
 13. A kit comprising: a) atleast one biologic sample collection device consisting of: soluble andhydrophilic polycaprolactone coupled to a carrier, wherein thepolycaprolactone is optionally coupled to one or more of a carbohydratetrehalose derivative group and at least one protein, and the carrieroptionally comprises one or more of an identifying label, aradio-frequency identification tag and a bar code, and wherein at leasta portion of the polycaprolactone solubilizes when exposed to a nucleicacid extraction reagent; and b) packaging.
 14. The kit of claim 13,further comprising instructions for use and at least one selected fromthe group consisting of: a nucleic acid extraction reagent; a nucleicacid labeling reagent; and a set of reagents for nucleic acidamplification.
 15. The kit of claim 13, wherein the packaging comprisesa polymer laminate or plastic container.
 16. The kit of claim 13,wherein the kit comprises a plurality of biologic sample collectiondevices.
 17. The kit of claim 16, wherein each biologic samplecollection device of the plurality of biologic sample collection devicesis individually packaged.
 18. The kit of claim 13, wherein the packagingcomprises: a first container comprising a rigid housing and a cap towhich the biologic sample collection device is attached, the biologicsample collection device positioned in the interior of the housing suchthat when the cap is operably attached to the housing, the biologicalsample collection device does not contact the interior of the housing;and a second container in which the first container and the biologicsample collection device are contained.
 19. The kit of claim 18, whereinthe first container is plastic and the second container is a plasticbag.
 20. The kit of claim 13, wherein the polycaprolactone is containedwithin at least one aperture disposed in the carrier.
 21. The kit ofclaim 20, wherein the polycaprolactone is contained within two or moreapertures disposed in the carrier.
 22. The kit of claim 20, wherein theat least one aperture is disposed at a first end of the carrier, and anidentifying label is disposed at a second end of the carrier.
 23. Thekit of claim 13, wherein the polycaprolactone is copolymerized with atleast one agent selected from the group consisting of: an acrylamide anda polyester other than polycaprolactone.
 24. The kit of claim 23,wherein the at least one agent is selected from the group consisting of:polylactide, polyglycolide, polydioxanone, acrylamide, polyN-isopropylacrylamide, and polyurethane.
 25. The kit of claim 13,wherein the biologic sample collection device has been sterilized. 26.The kit of claim 25, wherein the polycaprolactone has been treated witha base having a pH greater than 8 and a neutralizing agent forincreasing hydrophilicity of the polycaprolactone.
 27. The kit of claim26, wherein the base is selected from the group consisting of: NaOH,NaHCO₃, KOH, Na₂CO₃, and CA(OH)₂.
 28. A method of producing the biologicsample collection device of claim 1, the method comprising the steps of:providing a solution of solubilized polycaprolactone; submersing acarrier at least once into the solubilized polycaprolactone solutionsuch that the solubilized polycaprolactone solution coats the carrier;subjecting the solubilized polycaprolactone solution-coated carrier totreatment with a base having a pH greater than 8 and a neutralizingagent under conditions that result in soluble and hydrophilicpolycaprolactone coupled to the carrier; and sterilizing the soluble andhydrophilic polycaprolactone-coupled carrier such that the soluble andhydrophilic polycaprolactone-coupled carrier is free of nucleic acids,resulting in the biologic sample collection device of claim 1 comprisingsoluble and hydrophilic polycaprolactone coupled to the carrier, whereinat least a portion of the polycaprolactone solubilizes when exposed to anucleic acid extraction reagent.
 29. The method of claim 28, wherein thecarrier is submersed into the solution of solubilized polycaprolactoneusing a fabricated mold.
 30. The method of claim 28, wherein the base isselected from the group consisting of: NaOH, NaHCO₃, KOH, Na₂CO₃, andCA(OH)₂.
 31. The method of claim 28, wherein sterilizing the soluble andhydrophilic polycaprolactone-coupled carrier and rendering it free ofnucleic acids comprises exposing the soluble and hydrophilicpolycaprolactone-coupled carrier to UV radiation.
 32. The method ofclaim 28, further comprising copolymerizing the polycaprolactone with atleast one agent selected from the group consisting of: an acrylamide anda polyester other than polycaprolactone.
 33. The method of claim 28,further comprising adhering to or coupling to the carrier at least oneselected from the group consisting of: a radio-frequency identificationtag, a bar code, and an identification label.
 34. A method of extractingnucleic acids from a biologic sample collected by the biologic samplecollection device of claim 1, the method comprising the steps of:contacting the biologic sample and the biologic sample collection devicewith at least one nucleic acid extraction solution under conditions suchthat the polycaprolactone is solubilized or dissolved, resulting in asolution comprising nucleic acids separated from the biologic samplecollection device; and extracting the nucleic acids from the solution.35. The method of claim 34, wherein the step of contacting the biologicsample and the biologic sample collection device with at least onenucleic acid extraction solution comprises immersing the biologic sampleand the polycaprolactone in the at least one nucleic acid extractionsolution.
 36. The method of claim 34, wherein the at least one nucleicacid extraction solution comprises a proteolytic enzyme.
 37. The methodof claim 34, wherein the biologic sample is a human buccal sample. 38.The method of claim 34, wherein about 50% to about 95% of the nucleicacids from the biological sample are extracted from the biologicalsample.
 39. The method of claim 34, wherein the carrier comprises aradio-frequency identification tag and the method further comprisesobtaining information from the radio-frequency identification tag,wherein the information is at least one selected from the groupconsisting of: date, time, person, location, collector, and case number.40. A method for collecting a biologic sample, the method comprising thesteps of: contacting the biologic sample collection device of claim 1with a biologic sample such that the biologic sample is reversiblyadhered to the polycaprolactone; contacting the biologic samplecollection device and the biologic sample with at least one nucleic acidextraction reagent under conditions such that the polycaprolactone issolubilized and the sample is separated from the biologic samplecollection device; and collecting the separated sample.
 41. The methodof claim 40, further comprising extracting nucleic acids from theseparated sample.
 42. The method of claim 41, wherein the nucleic acidsconsist of deoxyribonucleic acid.
 43. A method of producing the biologicsample collection device of claim 1, the method comprising the steps of:providing a solution of solubilized polycaprolactone; forming at least afirst flat sheet of solubilized polycaprolactone; subjecting the atleast first flat sheet of solubilized polycaprolactone to treatment witha base having a pH greater than 8 and a neutralizing agent underconditions that result in soluble and hydrophilic polycaprolactone thatsolubilizes when exposed to a nucleic acid extraction reagent; couplingthe at least first sheet of soluble and hydrophilic polycaprolactone toa carrier; and sterilizing the soluble and hydrophilicpolycaprolactone-coupled carrier such that the soluble and hydrophilicpolycaprolactone-coupled carrier is free of nucleic acids, resulting ina biologic sample collection device comprising soluble and hydrophilicpolycaprolactone coupled to the carrier, wherein the soluble andhydrophilic polycaprolactone is a flat sheet, and at least a portion ofthe soluble and hydrophilic polycaprolactone solubilizes when exposed toa nucleic acid extraction reagent.